# Monomeric Glycine oxidase from Azotobacter vinelandii for Glycine biosensing

## TL;DR

This paper introduces a new monomeric glycine oxidase from Azotobacter vinelandii for use in low-cost, fast glycine biosensors that can detect glycine in bodily fluids like urine and sweat.

## Contribution

The study characterizes a novel monomeric glycine oxidase from Azotobacter vinelandii suitable for biosensing applications.

## Key findings

- The enzyme was confirmed to be monomeric, which is favorable for biosensor immobilization.
- Biosensors using this enzyme showed linear detection ranges of 0.1 to 3 mM and 0.1 to 5 mM.
- The enzyme exhibited substrate inhibition with kinetic parameters Km=4.65, kcat=0.38, and ksi=23.0.

## Abstract

Glycine level is an indicator of diseases related to the central nervous system and metabolic disorders such as diabetes and obesity. It is, therefore, relevant to have tools that can detect glycine at physiological concentrations. Biosensors are a reliable alternative to chemical methods for glycine detection, with low cost and high response speed. In the case of glycine detection, a glycine oxidase (GO) can be used as the molecular recognition element in a biosensor. The GO from Azotobacter vinelandii is an uncharacterized enzyme with high sequence identity to the only monomeric FAD-dependent GO from P. putida KT2440. The monomeric state is favorable for oriented immobilization of active sites in a biosensor, which is attractive for future applications. This work characterized the GO from A. vinelandii, and a second-generation glycine amperometric biosensor was assembled. The enzyme was recombinantly expressed and purified. The oligomeric state of the enzyme was also confirmed as the monomer. Therefore, a substrate inhibition behavior was detected in the enzyme kinetics experiments with parameters Km=4.65, kcat=0.38, and ksi=23.0. Two second-generation biosensors were assembled, and electrochemical characterization for the detection of glycine was carried out by chronoamperometry. The linear ranges of the biosensors (0.1 to 3 mM and 0.1 to 5 mM, for one- or two-enzyme layers respectively) are in the range of concentration of physiological fluids such as urine and sweat. This research opens new opportunities for applying new strategies that involve monomeric FAD-dependent glycine oxidases in glycine biosensing.

The online version contains supplementary material available at 10.1007/s11274-025-04657-4.